In accordance with RFA-AG-08-003, our long term goal is to define juvenile protective factors that protect the endothelium in the young, but are lost or altered with aging, and further compromised in the presence of genetic susceptibility to cardio- and cerebro-vascular disease. Emerging research data and our own preliminary data pinpoint two candidate juvenile protective factors: 1) endothelial progenitor cells (EPCs) and 2) platelets (plts). While their normal interactions suggest a pathway for synergistic repair mechanisms on the one hand, their dysfunctional interactions delineate a pathway for synergistic pathogenic mechanisms. Capitalizing on our transgenic rat model of coronary atherosclerosis with male susceptibility, and rat stroke model with increased female-susceptibility, and validated experimental tools and technologies, we prioritize the following Aims in the investigation of EPCs and platelets as two interacting candidate juvenile protective factors. Aim 1. Identification of EPC- and platelet-characteristics that define them as "juvenile protective factors", the time point at which the phenotype switch to "aged EPCs" occur, and the impact gender and disease susceptibility have on the "juvenile protective cellular and functional profile" of EPCs and platelets. Aim 2. Identify gene-networks involved in the switch in EPCs and platelets from 'juvenile protective factors'to 'aging-deficient factors', in order to pinpoint targets for "rejuvenation of aged EPCs" and address feasibility of "rejuvenation and subsequent transfusion" of autologous EPCs. This will be done by determining gene- pathway changes associated with maturational and aging changes in EPCs and platelets by targeted comparative proteomics using multiple reactions monitoring, and by signaling antibody (ab)-array profiling assessing constitutive changes in signaling molecules. Targeted pathway analysis will focus on gene networks involved in EPC-mediated endothelial repair and EPC-platelet interactions, and those implicated in EPC dysfunction. Aim 3. Provide proof of concept for juvenile protective factor-based intervention for cardiovascular and cerebrovascular disease by determining effects of in vivo transfusion of juvenile EPCs and platelets on disease progression of coronary heart disease and stroke in validated transgenic rat models of CAD (Tg25- cad) and stroke (Tg25-sp). Proposed studies will provide insight into i) the roles of EPCs, platelets and EPC- platelet complexes as candidate juvenile protective factors, ii) identify pathways for "rejuvenation of aged EPCs and/or platelets", and importantly, iii) provide proof-of-concept of juvenile-factor based intervention efficacy in the maintenance/repair of vascular endothelium. Information to be obtained will provide insight into the roles of endothelial repair mechanisms in attenuating cardiovascular and cerebrovascular disease mechanisms and target organ complications. We propose to study endothelial progenitor cells (EPCs) and EPC-platelet complexes as candidate juvenile protective factors which are critical for endothelial health but are lost with aging, and in heart disease and stroke. Through the use of transgenic rat models of heart disease and stroke, we will investigate how EPCs are altered with age, by sex-specific differences, and by genetic susceptibility to hypertension, heart disease and stroke in terms of their cell subtypes, proteome, signaling cascades. Knowing how they differ, we will then transfuse best candidates to see if they can alter hypertension, heart disease or stroke in corresponding rat models, thereby providing proof of concept for future clinical studies.